Inducible nitric oxide synthase expression by peritoneal macrophages in endometriosis- associated infertility  Barbara H Osborn, M.D., A.F Haney, M.D., Mary A Misukonis, B.S., J.Brice Weinberg, M.D.  Fertility and Sterility  Volume 77, Issue 1, Pages 46-51 (January 2002) DOI: 10.1016/S0015-0282(01)02940-5

FIGURE 1 Total peritoneal fluid NOx content and cell number. Total NO content of peritoneal fluid (NOx = NO concentration times volume) was highly correlated (r = 0.88, P=.002) with total peritoneal cell number in women with endometriosis, but not in normal fertile women. Osborn. Nitric oxide and endometriosis. Fertil Steril 2002. Fertility and Sterility 2002 77, 46-51DOI: (10.1016/S0015-0282(01)02940-5)

FIGURE 2 NOS activity and NOS2 antigen content of freshly isolated peritoneal macrophages. Upper panel: Peritoneal macrophage extracts from freshly isolated, non-cultured cells were analyzed for NOS enzyme activity (n = 9 for endometriosis, and n = 7 for normal). Mean activity was significantly higher in women with endometriosis than in normal fertile women (P=.0095, Wilcoxon rank-sum test). In the box plot: bar = mean, • = median, box = 25th to 75th percentiles, whiskers = 10th and 90th percentile. Lower panel: Immunoblots were performed on protein extracts of freshly isolated peritoneal macrophages. Positive controls for NOS2 were J7741 cells (J+) and cytokine activated DLD-1 cells (D+), cells that both express NOS2. Dark bands at approximately 133 kd, representing NOS2, were present in all five women with endometriosis (E1–E4, one not shown in figure), while none of the cells from the four normal women (N1–N4) had detectable NOS2 antigen (P=.008, Fisher’s exact test). Osborn. Nitric oxide and endometriosis. Fertil Steril 2002. Fertility and Sterility 2002 77, 46-51DOI: (10.1016/S0015-0282(01)02940-5)

FIGURE 3 NO production by and NOS activity in peritoneal macrophages after three days of culture in vitro. Peritoneal macrophages from women with endometriosis (n = 6) and normal fertile women (n = 3) were cultured for 3 days. NOx (displayed as μM) represents the concentration of NO in the supernatant medium of the culture wells. Cells from women with endometriosis had significant increases in NO production when cultured with IFN-α (P=.028, Wilcoxon signed-ranks test) or IFN-γ + LPS (P=.028), while those from normal fertile women did not. Cells from endometriosis patients produced more NO than those from normal women when IFN-α (P=.02, Wilcoxon rank-sum test) or IFN-γ + LPS (P=.039) were added. NOS enzyme activity (displayed as pmol L-citrulline/mg protein/100) was measured on cellular extracts of macrophages cultured for 3 days in culture. A significant increase in NOS activity occurred when IFN-α (P=.028, Wilcoxon signed-ranks test) or IFN-γ + LPS (P=.046) was added to cells from women with endometriosis, but not cells from normal fertile women. When compared to cells from normal fertile women, NOS activity was significantly higher in cells from endometriosis patients when cultured without additive (P=.02, Wilcoxon rank-sum test) or with IFN-α (P=.02), but was not different when IFN-γ + LPS was added. Osborn. Nitric oxide and endometriosis. Fertil Steril 2002. Fertility and Sterility 2002 77, 46-51DOI: (10.1016/S0015-0282(01)02940-5)